The main objectives of this project are the elucidation of the cellular functions of the family of ADP-ribosylation factors (ARFs) and the mechanism of their regulation. As Brefeldin A (BFA) has recently been shown to affect ARF functions and cellular distribution, we have recently expanded our efforts to include a better understanding of the mechanism of BFA action. Specific inhibitors of ARF activities have been exploited to provide evidence for the role of ARF proteins in a variety of intracellular membrane events. Anti-ARF peptides were found to be potent inhibitors of ER-Golgi transport, intra-Golgi transport, and endosome-endosome fusion. In addition, sensitivity of each of these assays, as well as nuclear membrane fusion, to GTPgammaS was shown to be mediated by ARF proteins. A search for regulators of ARF activity has identified an activity on Golgi membranes which appears to activate ARF. This ARF activating protein (AAP) is being investigated as a potential site of action of Brefeldin A and for its potential role as regulator of the protein secretory pathway. The ability of Brefeldin A to alter the structure of the Golgi and location of specific coat proteins (ARF and Beta-COP) is being investigated in concert with the development of Brefeldin A as a potential anti-cancer agent. A protein with the ability to specifically bind ARF and promote the GTPase activity by ARF1 has also been identified and is partially purified. This ARF-GAP (GTPase activating protein) is under investigation as both potential effector and down-regulator of ARF actions.